Preparation of cobalamins



All8 8, 1961 D. PERLMAN 2,995,498

PREPARATION oF ooBALAMINs med July 17, 1957 DAVID PERLMAN 3mm United States Patent() 2,995,498 l PREPARATION F -CQBALAMINS David Perlman, Princeton, NJ., assiguor to Olin Matlueson Chemical Corporation, New York, N.Y., a corporation of Virginia Filed July 17, 1957, Ser. No. 672,485

Claims. (Cl. 195-114) This application is a continuation-impart of my parent applications, Serial Nos. 604,248, 613,341, now Patent No. 2,842,540, 628,191, now Patent No. 2,892,443, and 635,298, now Patent No. 2,870,444, filed August 15, 1956, October 1, 1956, December 13, 1956, and January 22, 1957, respectively.

This invention relates to an improved process for preparing physiologically active cobalamins and more particularly to the preparation of vitamin B12 and other unnatural cobalamins possessing vitamin B12-'like activity by culturing a precursor-requiring vitamin B12-producing rnicroorganism of the genus Propionibacterium under substantially anaerobic conditions in a precursor-containing nutrient medium, and recovering the resultingphysiologically active cobalamin.

This process alfords a method whereby avariety of physiologically active cobalamins can be prepared ernploying the same microorganism and nutrients by varying only the precursor. [By the term precursor is meant a chemical substance which is utilized by the cobalaminforming rnicroorganism in the biosynthesis of the base of the nucleotide of the cobalamin, and which may be exempliiied by 5,6-dimethylbenzimidazol, which is a precursor for vitamin Bm] Among the microorganisms which may be employed in the practice of this invention are those Propionibacteria which Iare known to produce vitamin Bmin the presence of a B12-precursor such as 5,6-dimethylbenzimidazol. These include Proponbacterium arabnosum, P. thoemi, P rubrum, P. zeae, P. pentosaceum and P. petersoni.

The nutrient media useful in the process of this invention include the usual sources of assimilable carbon and nitrogen. As sources of assimilable carbon, there may be used: -(1) carbohydrates such as glucose, fructose,

xylose, galactose, lactose, and maltose; (2) substances' containing carbohydrates such as whey, milk, cornsteep liquor, grain mashes, and molasses; (3) polyhydric alcohols such as glycerol and mannitol; (4) fats, such as lard oil, soybean oil, corn oil, butterfat and cotton seed oil; and (5) fatty acids such as acetic propionic, pyruvic, stearic, palmitic, oleic, and linoleic. Sources of available nitrogen include: (l) organicnitrogen compounds such as proteinaceous materials e.g. casein, urea, soybean meal, sh meal, yeast or yeast products, whey or whey concentrates, amino acids and liver cake; and (2) inorganic compounds such as nitrates or ammonium compounds.v The nutrient media should also include metallic 2,995,498 Patented Aug. 8, i961 cations including cobalt, molybdenum, potassium, sodium, magnesium, iron, copper, manganese, the anions chloride, sulfate, phosphate, and carbonate, and the vitamins thiamin, niacin, biotin, folic acid, pyridoxine, riboflavin, and p-aminobenzoic acid. These ions and vitamins may be present in the crude materials used in the nutrient medium.

The fermentation process may be carried out at temperatures from about 20 C. to about 40 C. and may be operated under essentially anaerobic or microaerophilic conditions. The precursor (leading to the formation of Y in the drawing) may be added to the fermenting culture either continuously or intermittently during the incubation period. After a sulicient incubation time (about one to ten days), the fermented medium may be dried and incorporated as a supplement into feeds fed to chickens and pigs, or the cobalamin present may be recovered in purified form, either as a hydroxocobalamin or, after treatment with potassium cyanide or other cyanide containing salts, as the cyano-cobalamin as more fully detailed in the examples following. Ifacyanocobalamin is recovered, it can be converted to hydroxocobalamin and thence to other salts -by methods known in the a-rt [see Kaczka et al., Jour. Amer. Chem. Soc., 73,

3569 1951)] to yield products which are also biologically active.

'Ihe cobalamins prepared by the process of this invention may be represented by the structural formula in the drawing, wherein X is a hydroxy radical or an anion, preferably the anion of a phannacologically-acceptable acid. Examples of such anions are the anions of the mineral acids (e.g. chloride, bromide, sulfate, sulte, nitrate and nitrite), cyanide, and cyanate. The compounds prepared by the process of this invention can thus be termed Y-X-cobalamins [using the nomenclature of Bernhauer et al., Ari-gew. Chemie, 66, 776 (l954)], where Y represents the base present in the nucleotide portion of the cobalamin molecule and X represents the anion. The cobalarnins synthesized by the process of this invention are biologically active, and hen can be used in lien of vitamin B1, (5,6-dimethylbenzimidazolecyano-cobalarnin) in promoting growth of chicks. For this purpose they are administered in the same manner as vitamin B12 [e.g. as supplements to chick feeds, or by injection as described by Coates et al. Biochem. I. 64, 682 (1956)] the respective dose of the particular cobalamin depending on its potency relative to vitamin B12.

The nature of the precursor employed depends on the physiologically active cobalamin desired. Thus, if vitamin B13 is desired, 5,6-dimethylbenzimidazole or another vitamin B12 precursor, such as 2,3dimethyl5,6diamino benzene, 2,3-dinitro-5,6-dimethy1benzene or 2,3-dimethyl- 4-amino-5-nitrobenzene, may be used. Thus, as summarized in the following table, the nature of the resulting cobalamin will depend on the precursor chosen. In this table, the symbol Y is that in the accompanying drawing.

Product (Y -eobalamins) Name Product (Y-eobalamlns) Precursor Name Yis- I Phnnavinn Phenzineebalamn \N I 1I 2nitrophenazine 2-nitrophenazine-eobalamin NO,

2aminophenazine zaminophenazine-oobalamin NH,

N'Hg 1I Lanaino-2-hydroxy-phenazine 1amlno2hydroxyphenazineoobalamm 0H NHcooH. Il l-aoetamido-B-methoxyphenazine 1aoetamido-3-methoxyphenazine-cobalamin OCHs N I I If 2-hydroxyphenazine 2-hydroxyphenazine-cobalamln (l OH I N\ /CH Z C C-CH: -methylbenzmidazole 6-methy1benzimidazo1e-cobalamin C H \Q/ \CH I Quinoxaline Quinoxallne-cobalamin /N /N Benzotrazole Benzotriazole-cobalamin N/ To show the homogeneity and activity of the cyanopaper strip of Whatman 3 mm. paper in parallel with cobalamins formed in the examples of this invention, the samples of 5,6-dimethylbenzimidazole-cyanocobalamin, following tests were conducted. For the tests the co- 70 adenine-cyanocoba1amin,'2 -methyl-adenine-cyanocobalabalamin was dissolved in water to give -a concentration of min and Fords factor B [Ford et al., Biochem. Jour.,

between 10 and 100 micrograms per ml. of Water: 59, 86 1955 The sheet is placed in an ionophoresis TEST I apparatus [similar to that described by Holdsworth in Nature, 171, 148 (1953)], and the paper is impregnated The solution of the cyanocobalamin is dried on a filter 75 with a solution of 0.5 N acetic acid containing 0.02%

KCN (W./v.). A potential of `about 280 'volts is applied for about 17 hours. The sheet is removed and dried. When dry (and free from odor of acetic acid), it is applied for 15 minutes to the surface of an agar plate seeded with a suspension of a vitamin B12-requiring strain of Escherichia coli (ATCC 11105). [The agar medium contains (grams/liter): sucrose, 20 g.; citric acid, 1.2 g.; (NH4)2HPO4, 0.4 g.; KCl, 0.08 g.; MgClgHgO, 0.418 g.; MnCl24H2O, 0.036 g.; FeCl36H2O, 0.023 g.; ZnClg, 0.021 g.; CoCl3-6H2O, 0.04 g.; agar 15 g.; triphenyl tetrazolium chloride, 1 g.] After 18 hours incubation at 37 C., the agar plate is observed. The positions of zones of lgrowth of bacteria (noted as red zones due to the reduction of the tetrazolium dye to the colored formazan) are noted in relation to the location on the paper strip where the samples are applied. The results obtained are recorded in the examples.

TEST II An aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatman No. l filter paper parallel to spots of known cobalamins. The chromatogram is developed lby the descending method using a Solvent mixture containing: 77 ml. of sec-butanol, 23 m1. of water, 0.25 rnl. of KCN solution (5 gms./ 100 ml.) and 100 mg. of KClO4 for 24 hours (at 25 C.). The strip is dried and applied to the seeded agar plate as in Test 1. After incubation,.the zones of growth, representing the presence of vitamins of the B12 group (measured with reference to the movement of 5,6-dimethylbenzimidazole-cyanocobalamin), are determined.

TEST III (a) An aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatman No. 4 filter paper parallel to spots of known cobalamins. The chromatogram is developed by the descending method using a solvent mixture containing: sec-butanol, 100 ml.; water, 50 ml.; KCN [5% solution (w./v.)], 0.25 ml.; 'and NH4OH (concentrated), 1.0 ml. After 17 hours development (at 35 C.), the strips are dried and plated on seeded agar plates as in Test I. Zones of growth are determined.

(b) Same as Test Illa with 1.0 ml. of glacial acetic acid substituted for the ammonium hydroxide.

TEST IV An aliquot is assayed for the presence of substances stimulating the growth of Lactobacillus lechmanni (ATCC 7830) using as standard 5,6-dimethylbenzimidazole-cyanocobaiamin and the method of the U.S. Pharmacopeia (15th edition). A value is determined.

TESTV An aliquot is assayed by the method of Ford and Porter [Brit. I. Nutrition, 7, 326 (1953)], using the growth response of Ochromonas malhamensis and 5,6-dimethylbenzimidazole-cyanooobalamin as standard. A value is obtained.

When the cobalamins listed hereinbefore in the form of the cyano salts (X=CN) are analyzed by these tests, it was found that each of the cobalamins was active in Tests IV and V and the following additional results were obtained.

Behavior of cobalamins with reference to 5,6-drnethylbenzimidazole-cyano-cobalamin Cobalamln Test I Test Test Test II 1 lIIs 1 IIIbl 6methylbenzamidazole---. Ionophoretically 0. 88 1. 0 1. 0

neutral.

Benzimidazolo-... 0.90 0.95 0.95

Benzotrlazole--. 1. 05 0.3 1. 0

2,3-naphthimldazol 1. 0 l. 0 l. 0

5-triluoromethylbenzimid 0. 95 0. 95 0. 95

BZ() G.

4-bromo-6-methoxy-ben- 0. 74 0. 78 1. 0

zlmldazole.

2,4-dlchloroquinazollne--. 0. 1 0. 9 0. 6

1,2,3,4 tetrahydro 2,4 di- 0. 37 0. 39 0. 23

oxoqulnazoline.

8-amlno-4 methoxy quin- 0. 4 0. 42 0. 28

azoline.

2 methyl 4 methylthio Same as adenine 0. 48 0. 64 0. 28

quinazollne. cobalamln.

Phenazine Ionophoretically 0. 5 0. 6 0. 35

neutral.

2-aminophenazlne do 0.8 0. 97 1. 0

l-alnlinod-hydroxyphen- -....do 0.3 0.21 0.6

az e.

1-acetamldo-3 -methoxy- ....-do 0.13 1.05 0.66

phenazlne.

Quinoxaline do 0.86 0. 7 0.9

l Mobility relative to 5,6-dimethylbenzlmidazole-cyano-cobalarnln.

The following examples illustrate the invention (all temperatures being in centigrade):

EXAMPLE 1 Vifamin B13 A medium containing 30 g. glucose, 20 g. autolyzed yeast, 0.01 g. Co(NO3)z-6H2O, and tap water to l liter is prepared and 500 ml. aliquots are added to 1 liter Erlenmeyer asks. The asks are plugged with nonabsorbent cotton and autoclaved at 121 for 30 minutes. When the flasks have cooled to room temperature, l0 g. of heat sterilized powdered calcium carbonate is added. The medium is then inoculated with 5 to l0 ml. of a culture of Propionbacterium arabnosum A.T.C.C. 4965 (American Type Culture Collection, Washington, D.C.) which has grown on this medium for 72 hours. The asks are then placed on a reciprocating shaker (120 l-inch strokes per minute) located in a room maintained at 30. A sterile alcoholic solution of 5,6-dimethylbenzimidazole is prepared by dissolving 50 mg. of the 5,6-dimethylbenzimidazole in 10 ml. of 70% v(v./v.) aqueous ethanol and filtering the solution through a sterile fritted ultra line glass filter. One ml. of the solution is added to the inoculated medium (after inoculation) and the supplementation is repeated at 24-hour intervals during the ensuing incubation period. 'I'he culture is allowed to grow in the medium and small aliquots are removed daily for pH measurement. When the pH falls below pH 6 (usually on the second day of incubation) l0 ml. of 2 N NaOH are added to adjust the pH to 6.8 to 7.2; this neutralization is repeated daily if the fermented medium is below pH 6. A solution of glucose is added after 3 days incubation so that the concentration of glucose in the fermenting medium is between 30 and 40 mg. per ml. (a sterile concentrated glucose solution containing 50 grams of glucose per ml. is convenient for this purpose). After 6 days incubation, an aliquot is removed, treated, and analyzed as described above.

Instead of adding the 5,6-dimethylbenzimida2ole solution once a day, continuous addition may be used with the concentration of the added solution adjusted and rates of addition controlled so that between 5 and 200 mg. of the benzimidazole are added per liter of medium during the incubation period. Compounds which may be substituted for the 5,6-dimethylbenzimidazole and yield 5,6-dimethylbenzimidazole-cobalamin are listed in Table I.

EXAMPLE 2 trifluoromethyl-benzimidazole-cyano-cobalamin 30 liters of a medium containing (per liter): glucose, 30 g.; autolyzed yeast, 20 g.; Co(NO3)2-6H2O, 0.01 g.; tap water, 1 liter; are placed ina stainless steel fermentation unit of 38 liters capacity, heated at 121 for 30 minutes, and cooled to 30. About 2,000 ml. of a slurry of CaCO3 (containing 600 g. of CaCOa), sterilized by autoclaving, is then added together with l liter of Propionibacterium arabinosum A.T.C.C. 4965 culture grown on this medium for 72 hours in fiasks shaken on a reciprocating shaker (120 l-inch strokes per minute), located in a 30 constant temperature room. A sterile alcoholic solution of 2-nitro-4trifluoromethylaniline (sterilized by filtration through a fn'tted glass filter) containing 1 mg. per ml. is then added so that the final concentration of aniline is 5 mg. per liter (this supplementation being repeated at 24-hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is 30 g. per liter. After 5 days of incubation, the pH of the mixture is about 5.3. The

fermented medium is then passed through a Sharples Super Centrifuge, or the cells containing 5-triuoromethylbenzimidazole-hydroxo-cobalamin are separated by filtration through a bed of filter-aid (Celite).

The collected cells are resuspended in 3 liters of propanol-water (80:20) containing 0.1 g./liter of potas sium cyanide, and the suspension is heated at 80-90 for 20 minutes. After filtration, the filtrate is concentrated to about 750 ml. The pH is adjusted with N sodium hydroxide to about 7.5, and 25 g. of activated carbon (Darco G60) is added and the mixture stirred. The activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (65:35 V./v.), containing 0.1 g./liter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three equal volumes of phenol-benzene (70 parts 88% phenol-30 parts benzene). The non-aqueous layer is separated, pooled and diluted with benzene so that the ratio of benzene to phenol is l. The solution is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concentrated to about 50 m1. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the rich-water is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous llayer is reextracted with onetenth its volume of benzyl alcohol. The rich benzyl alcohol extracts are combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column is washed with 1 part` of methanol and 2 parts of acetone. The rich material is eluted with methanol and the eluate dried under vacuum. The residue is dissolved in a minimum of water and acetone is added until slightly turbid. On standing for several days, 5-triuoromethyl-benzimidazole-cyano-cobalamin in the form of red needlelike crystals arerecovered. Spectroscopic examination shows maxima at 278, 361, 520 and 5 50 my.

[Etta 204 (361 mil EXAMPLE 3 5-Irfluor0methyl-benzmdazole-hydroxo-cobalamin To a solution of 10 mg. of S-trifiuoromethyl-benzimidazolecyanocobalamin in 10 ml. of water is added 50 mg. of platinum oxide catalyst, and the mixture is shaken with hydrogen at atmospheric pressure for 24 hours. The filtrate from the catalyst is evaporated in vacuo at 25. The residue is dissolved in 1 ml. of water and 12 m1. of acetone are added. Dark red crystals of 5-triuoromethy1-benzimidazole-hydroxocobalamin are l0 found on standing for several days. Spectroscopie examination shows maxima at 315, 352, 415 and 530 mp.

[EPL 170 (352 mp)] EXAMPLE 4 5trifiuoromethyl-benzimdazoIe-chloro-cobalamin 10 m. of S-triliuoromethyl-benzimidazole-hydroxo-co-. I

balamin is dissolved in 3 ml. of water and the solution adjusted to pH 4 with 0.1 N hydrochloric acid. The solution is then diluted with 10 ml. of acetone, and after a short time the chloro-cobalamin begins to crystallize in the form of dark red needles. The crystals are separated by centrifuging, washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium nitrite and acetic acid, potassium cyanate and dilute sulfurie acid for the hydrochloric acid in the procedure of Example 4, the corresponding bromo, nitro, cyanato and sulfatocobalamins, respectively, are obtained.

EXAMPLE 5 4-brom06methoxy-benzimdazole-cyano cobalamn Thirty liters of a medium containing `(per liter): glucose, 30 g.; autolyzed yeast, 20 g.; Co(NO3)-6H2O, 0.01 g.; tap water, 1 liter; are placed in a stainless steel fermentation unit of 38 liters capacity, heated at 121 for 30 minutes and cooled to 30. About 2,000 ml. of a slurry of CaCO3 (containing 600 g. of CaCOs), sterilized by autoclaving, is then added together with 1 liter of Propionbacterium arabinosum A.T.C.C. 4965 culture grown on this medium for 72 hours in flasks shaken on a reciprocating shaker (120 l-inch strokes per minute), located in a 30 constant temperature room. A sterile aqueousalcoholic solution of 3-bromo-5methoxyphenylenedia mine-1,2 (sterilized by filtration through a fritted glass filter) containing 1 mg. per ml. is then added so that the final concentration of added compound is 5 mg. per liter (this supplementation being repeated at 24-hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is30 g. per liter. After 5 days of incubation, the pH of the mixture is about 5.3. The fermented medium is then passed through a Shar-ples Super Centerfuge, or the cells containing 4bromo6methoxybenzimid azole-hydroxo-cobalamin are separated by filtration through a bed of filter-aid (Celite).

The collected cells are resuspended in 3 liters of propanol-water (80:20) containing l g./liter of potassium cyanide,.and'the suspension is heated at 80-90 for 20 'minutes After filtration, the filtrate is concentrated to about 750 ml. The pH is adjusted with 10 N sodium hydroxide to about 7.5, and 25 g. of activated carbon (Darco G60) is added and the mixture stirred. The activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (65:35 v./v.), containing 0.1 g./liter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three equal volumes of phenol-benzene (70 parts 88% phenol- 30 parts benzene). The non-aqueous layer is separated, pooled and diluted with benzene so that the ratio of benzene to phenol is 15:1. 'Ihe solution is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concentrated to about 50 ml. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the rich-water is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous layer is reextracted with onetenth its volume of benyzl alcohol. The rich benzyl alcoof acetone. The rich material is eluted with methanol and the eluate dried under vacuum. The residue is dis- -methoxy-benzimidazole-cyano-cobalamin in the form of red needlelike crystals are recovered. Spectroscopie examination shows maxima at 278, 361, 520 and 550 miv [Ella 204 (361 mfl EXAMPLE 6 4-bromo-6-methoxy-benzimidazole-hydroxo-cobalamn To a solution of mg. of 4-bromo-6-methoxy-benzimidazole-cyano-cobalamin in 10 ml. of water is added 50 mg. of platinum oxide catalyst, and the mixture is` shaken with hydrogen at atmospheric pressure for 24 hours; The filtrate from the catalyst is evaporated in vacuo at 25. and 12 ml. of acetone are added. Dark red crystals of 4 bromo-6-methoxy-benzimidazole-hydroxo-cobalamin,

are found on standing for several days. Spectroscopie examination shows maxima at 315, 352, 415 and 530 m/r,

cobalamin is dissolved in 3 m1. of water and the solution adjusted to pH 4 with 0.1 N hydrochloric acid. The

solution is then diluted with 10 ml. of acetone, and after The residue is dissolved in 1 ml. of water activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (65:35 v./v.), containing 0.1 g`./liter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three equal volumes of phenol-benzene (70 parts 88% phenol-30 parts benzene). The non-aqueous layer is separated, pooled and diluted with benzene so a short time the chlorocobalamin begins to crystallize in the form of dark red needles. The crystals are separated by centrifuging, washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium l nitrite and acetic acid, potassium cyanate and dilute sulfuric Iacid for the hydrochloric acid in the procedure of Example 7, the corresponding bromo, nitro, cyanato and sulfato-cobalamins, respectively, are obtained.

EXAMPLE 8 (3,4-dihydro-4-oxoqunazoln-I,3-diyl)-cyano-cobalamin Thirty liters of a medium containing (per liter): glu-'i cose, ig.; autolyzed yeast, 20 g.; Co(NO3)2-.6H2O, 0.01 g.; tap water, l liter; are placed in a stainless steel fermentation unit of 38 liters capacity, heated at 121 for 30 minutes and cooled to 30. About 2,000 ml. of a slurry of CaCO; (containing 600 g. of CaCO3), sterilized by autoclaving, is then added together with 1 liter of Proponibacterium arabnosum A.T.C.C. 4965 culture grown on this medium for 72 hours in flasks shaken on a reciprocating shaker (120 l-inch strokes per minute),

located in a 30 constant temperature room. A sterile aqueous-alcoholic solution of 4(3H) quinazolinone (sterilized by filtration through a fritted glass filter) containing 1 mg. per ml. is then added so that the final concentration of added compound is 5 mg. per liter (this supplementation being repeated at 24-hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is 30 g. per liter. After 5 days of incubation, the pH of the mixture is about 5.3. The fermented medium is then passed through a Sharples Super Cennifuge, or the cells containing (3,4 dihydro 4 oxoquinazolin 1,3 diyl) hydroxo-cobalamin are separated by filtration through a The pH is adjusted with l0 N sodium that the ratio of benzene to phenol is 15:1. The solution is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concentrated to about 50 ml. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the rich-water is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous layer is reextracteu with one-tenth its volume of benzyl alcohol. The rich benzyl alcohol extracts are combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column is washed with l part of methanol and 2 parts of acetone. The n'ch material is i eluted with methanol and the eluate dried under vacuum. The residue is dissolved in a minimum of water and ace- Itone is added until slightly turbid. On standing for sev eral days, (3,4-dihydro-4-oxoquinazoliu-1,3diyl)-cyano cobalamin in the form of red needlelike crystals is recovered. spectroscopic examination shows maxima at 278, 361, 520 Iand 550 mn [EmL 204 (361 mp.)]

The product, (3,4-dihydro-4-oxoquinazolin-1,3-diyl) cyano-cobalarnin, is readily obtained, under the same conditions, using Proponibacterum pentosaceum (A.T.C.C. Number 4875 or Propionibacterium petersom'i (A.T.C.C. Number 4870) in place of Proponbacterium arabnosum in Example 8.

EXAMPLE 9 To a solution of l0 mg. of (3,4-dihydro-4-oxoquinazolin-1,3diyl)cyanocobalamin in 10 ml. of water is added 50 mg. of platinum oxide catalyst, and the mixture is shaken with hydrogen at atmospheric pressure for 24 hours. The filtrate from the catalyst is evaporated in vacuo at 25. The residue is dissolved in l ml. of water and 12 ml. of acetone are added. Dark red crystals of 3,4 dihydro 4 oxoquinazolin 1,3 diyl) hydroxocobalamin on standing for several days. Spectroscopie examination shows maxima at 315, 352, 415 and 530 ma lEll... (352 m)] EXAMPLE 10 (3,4-dhydro4-oxoquinazoIn-I ,3 -dyl Chlor-cobalamin 10 mg. of (3,4 dihydro 4 oxoquinazolin 1,3- diyl)hydroxocobalamin is dissolval in 3 ml. of water and the solution adjusted to pH 4 with 0.1 N hydrochloric acid. The solution is then diluted with 10 ml. of ace tone, and after a short time the chloro-cobalamin begins to crystallize in the form of dark red needles. The crys tals are separated by centrifuging, washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium nitrite and acetic acid, potassium cyanate and dilute sulfuric acid for the hydrochloric acid in the procedure of Example 3, the corresponding bromo, nitro, cyanato and sulfato-cobalamins, respectively, are obtained.

EXAMPLE 11 Five hundred ml. aliquots of the medium described in Example 8 are placed in 1 liter Erlenmeyer asks. The asks are plugged with non-absorbent cotton and autoclaved for about 20 minutes at 121 C. When the liquid i 13 has cooled to room temperature, grams of CaCO3 powder (sterilized by heating in a 150 C. oven for 2 hours) are added. This medium is then inoculated with 10 ml.'

of a 72 hour old culture of P. arabnosum prepared as described in Example 8. To the inoculated medium is added 2 m1. of an alcoholic solution of a suitable quinazoline precursor so that the level of added quinazoline precursor is 10 mg. per liter of medium. This supplementation is repeated daily for 7 days. The flasks are then placed on a reciprocating shaker (l201-inch cycles per minute) located in an incubator maintained at 30 C. After 3 days incubation, 20 m1. of a sterile glucose solution (50 grams glucose per 100 ml. solution) are added to each ask. VAt the end ofthe 7 day incubation period the fermented medium (pH is about 5.4) is heated at 90-95 C. for 30 minutes and then centrifuged. Suflicient KCN is added to the supernatant liquid to give a concentration of 0.1 g. per liter. The liquid is then extracted thrice with 1/2 its volume of a phenol-benzene solution (70:30). The phenol-benzene layers are pooled and diluted with an equal volume of n-butanol. Distilled water equal to 1A@ the volume of the mixture is added, and the mixture shaken vigorously and then centrifuged.

When the precursor added to the fermentation is 2,4 dichloroquinazoline, the cobalamin formed, (2,4-dichloroquinazolin-l,3diyl)cyanocobalamin is ionophoretically neutral (at pH 2.5. It has a mobility of 0.9 that of 5,6 dimethylbenzimidazole-cyano-cobalamin in the filter paper chromatographic system containing sec-butanol, water, KCN, and acetic acid. In the system containing sec-butanol, water, KCN, and KClO4, the mobility is about 0.1 that of the 5,6 dimethylbenzimidazole-cyan-cobalamin.

When the precursor added to the fermentation is 4- chloro-8-nitroquinazoline, the cobalamin formed, (4- ch1oro-8-nitro-quinazo1in-1,3 diyl) cyano-cobalamin is ionophoretically neutral (at pH 2.5). It has a mobility equal to 5,6-dimethylbenzimidazole-cyano-cobalamin in the filter paper chromatographic system containing secbutanol, water, KCN and acetic acid. In the system containing sec-butanol, water, KCN and KC1O4 the mobility is about 0.9 that of the 5,-dimethylbenzimidazole-cyanocobalamin.

When the precursor added to the fermentation is 2(1), 4(3)-quinazolinedione, the cobalamin formed, (1,2,3,4 tetrahydro 2,4 dioxoquinazolin-l,3diyl)-cyano-cobalamin, is ionophoretically neutral (at pH 2.5 It has a mobility of about 0.4 that of 5,6-dimethylbenzimidazolecyano-cobalamin in the filter paper chromatographic system containing sec-butanol, water, KCN and acetic acid.

When the precursor added to the fermentation is 8- amino-4-methoxyquinazoline, the cobalamin formed, (8- amino4methoxyquinazolin 1,3-diy1)cyanocobalamin, is ionophoretically neutral (at 2.5). In the filter paper chromatographic test using the solvent system containing sec-butanol, water, KCN andammonium hydroxide it has a mobility 0.3 relative to that of 5,6-dimethylbenzimidazole-cyano-cobalamin.

When the precursor added to the fermentation is 2- ethyl-4(3H)quinazolinethione, the cobalamin formed, (3,4-dihydro-2-ethyl 4 thiono-quinazolin-l,3-diyl)cy ano-cobalamin is ionophoretically neutral (at pH 2.5). In the filter paper chromatographic test using the solvent system containing sec-butanol, water, KCN and KCIO., it has a mobility of 0.45 relative to that of 5,6-dimethylbenzimdazole-cyano-cobalamin.

When the precursor added to the fermentation is 2- methyl-4-methylthioquinazoline, the cobalamin formed, (2-methyl4methylthioquinazolin-l,'3-diyl)cyanocobala min, moves at the same rate as adenine cobalamin in the ionophoresis (pH 2.5). In the filter paper chromatographic test using the solvent system containing sec-butanol, water, KCN and acetic acid it has the mobility of 0.64 relative to that of 5,6-dimethyl-benzimidazole-cyano-co- KCN ammonium hydroxide it has a mobility of 0.28 relative to that of 5,6-dimethylbenzimidazole-cyano-cobalamin; and in the system containing sec-butanol, water, KCN and KC104 the mobility is about 0.48 that of 5,6- dimethylbenzirnidazole-cyano-cobalamin.

When the precursor added to the fermentation is 2,4- quinazolinedithiol, the cobalamin formed, (2,4-dithioquinazolin-1,3diyl)-cyano-cobalamin, moves at the same rate as adenine cobalamin in the ionophoresis (pH 2.5 It has a mobility of 0.24 that of 5,6-dimethylbenzimidazole-cyano-cobalamin in the filter paper chromatographic test using the solvent system,containing sec-butanol, water, KCN and aceticacid. In the system containing sec-butanol, water KCN, and ammonium hydroxide, the mobility is about 0.65 that of the 5,6-dimethy1benzimidazole-cyano-cobalamin. In the system containing secbutanol, water, KCN and KClO4, the mobility is about v0.36 that of the 5,6-dimethylbenzimidazole-cyanoco balamin. i

EXAMPLE 12 30 liters of a medium containing (per liter): glucose, 30 g.; autolyzed yeast, 20 g.; Co(NO3)2-6H2O, 0.01 g.; tap water, l liter; are placed in a stainless steel fermentation unit of 38 liter capacity, heated at 121 for 30 minutes, and cooled to 30. About 2,000 ml. of a slurry of CaCO3 (containing 600 g. of CaCOs), sterilized by autoclaving, is then added together with 1 liter of Propionibacterium arabinosum A.T.C.C. 4965 culture grown on this medium for 72 hours in flasks shaken on a recipro eating shaker (1201-inch strokes per minute), located in a 30 constant temperature room. A sterile alcoholic solution of phenazine (sterilized by filtration through a fritted glass filter) containing l mg. per ml. is then added so that the final concentration of the phenazine is 5 mg. per liter (this supplementation being repeated at 24-hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is 30 g. per liter. After 5 days of incubation, the pH of the mixture is about 5.3. The fermented medium is then passed through a Sharples Super Centrifuge, or the cells containing (phenazin-9,10diyl)-hydroxo-cobalamin are separated by filtration through a bed of filteraid (Celite).

The collected cells are resuspended in 3 liters of propanol-Water (80:20) containing 0.1 g./liter of potassium cyanide, and the suspension is heated at 80-90 for 20 minutes. After filtration, the filtrate is concentrated to about 750 m1. The pH is adjusted with 10 N sodium hydroxide to about 7.5, and 25 g. of activated carbon (Darco G60) is added and the mixture stirred. The activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (65:35 v./v.), containing 0.1 g./liter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three 400 ml. volumes of phenol-benzene (70 parts 88% phenol- 30 partsl benzene). The non-aqueous layer is separated, pooled and diluted with benzene so that the ratio of benzene to phenol is 15:1. The solution is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concentrated to about 50 ml. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the rich-water is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous layer is reextracted with one-tenth its volume of benzyl alcohol. The rich benzyl alcohol extracts are combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column is washed with 1 part of methanol and 2 parts balamin. In the system containing sec-butanol, water, of acetone. The rich material is eluted with methanol and the eluate dried under vacuum. The residue is dissolved in a minimum of water and acetone is added until slightly turbid. On standing for several days (phenazin9,10diyl)-cyano-cobalamin in the form of red needlelike crystals is recovered. Spectroscopie examination shows maxima at 361, 520 and 550 mw.

[EP-.. 204 (361 mul] The product (phenazin-9,10-diyl)cyanocobalamin is readily obtained, under the same conditions, using Propionibacterium pentosaceum (ATCC number 4875) or Propionbacterium petersonii (ATCC number 4870) in place of Propionibacterium arabnosum `in Example 12.

EXAMPLE 13 (Phenazin-ShIO-diyl) -hydroxo-cobalamin To a solution of 10 mg. of (phenazin-9,10'diyl) -cyanocobalamin in 10 ml. of water is added 50 mg. of platinum oxide catalyst, and the mixture is shaken with hydrogen at atmospheric pressure for 24 hours. The filtrate from the catalyst is evaporated in vacuo at 25. 'I'he residue is dissolved in 1 ml. of water and 12 ml. of acetone are added. Dark red crystals of (phenazin-9,10diyl) hydroxo-cobalamin are found on standing for several days. 4Spectroscopie examination shows maxima at 352, 415 and 520 mn [Elta 170 (352 m,.)]

EXAMPLE 14 (Phenazin-9,1 -diyl -chloro-cobalamin l0 mg. of (phenazin-9,10diyl)hydroxocobalamin is dissolved in 3mi. of water and the solution adjusted to pH 4 with 0.1 N hydrochloric acid. The solution is then diluted with l0 ml. of acetone, and after a short time the chloro-cobalamin begins to crystallize in the form of dark red needles. The crystals are separated by centrifuging, washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium nitrite and acetic acid, potassium cyanate, dilute phosphoric'and dilute sulfuric acid for the hydrochloric acid in the procedure of Example 14, the corresponding bromo, nitro, cyanato, phosphate and sulfatocobalamins, respectively, are obtained.

EXAMPLE 15 (Z-nitroph enazin-9,I 0-diyl) -cyano-cobalamin Aliquots of the autolyzed yeast-glucoseCo(NO3) 2- 6H2O-water medium described in Example l2 are placed in Erlenmeyer asks (500 ml. per lter flask is a suitable quantity). 'Ihe flasks are plugged with non-absorbent cotton and autoclaved for 20 minutes at 121 After coolingto room temperature, l0 grams of calcium carbonate powder (previously sterilized by autoclaving at 120 for 30 minutes and heating in a 150 oven for l hour) is added. 'I'he asks are inoculated with l0 m1. of a 72 hour culture of Proponbacterum arabz'nosum A.T.C.C. 4965 and placed on a reciprocating shaker (120 l-inch strokes per minute) located in an incubator maintained at 30. Two ml. of a sterile alcoholic solution of 2-nitrophenazinelis added so that the final concentration of the phenazne is 5 mg. per liter. This addition is repeated 6 times at 24 hour intervals. Twenty ml. of a solution containing 50 grams of glucose per 100 ml. is added after 3 days incubation. After 6 days incubation on the shaker the fermented medium is heated at 90-95 for 20 minutes. Ten

ml. of a KCN solution (5 g./100 ml.) are added per liter 16 The benzene extracts are pooled and diluted with an equal volume of n-butanol. A quantity of water equivalent to lfm the volume of the phenol-benzene-butanol solutio'n is added and the mixture shaken vigorously and centrifuged. The red colored aqueous layer is separated and analyzed by the tests described herein before.

When analyzed by the ionophoretic method (pH 2.5)

a cobalamin with ionophoretically neutral properties is found. When analyzed by the filter paper chromatographic method using the solvent system sec-butanol,

water, 50; 5% KCN, 0.25 ml. and glacial acetic acid,l i

EXAMPLE 1e (Z-aminophenazin9,10diyl) -cyano-cabalamin 'Ihe procedure and method of testing the aqueous extract described in Example 15 is repeated except that a molar equivalent of Z-aminophenazine is used instead of 2nitrophenazine- The cobalamin is found Ato be ionophoretically neutral (when the pH of the system is 2.5 When studied by -iilter paper chromatography using the system containing sec-butanol, water, KCN, and glacial acetic acid, the new cobalamin has a mobility of 0.45 relative to that of 5,6 dimethylbenzimidazole-cyanocobalamin. If the system containing sec-butanol, water, KCN, and KC104 is used, the mobility of new cobalamin is 0.28 of that observed when 5,6-dimethylbenzimidazolecyano-cobalamin is added. 'I'he new cobalamin stimulates the growth of Ochromonas malhamenss.

EXAMPLE 17 l (I-amino-Z-hydroxy-phenazn-9J0-dyl) -cyano-cobalamn water, KCN, and acetic acid for developing solvent, the l has a mobility 0.21 that of 5,6-dimethy1benzimidazolecyano-cobalamin. 'Ihe new cobalamin stimulates the growth of Ochromonas malhamenss.

EXAMPLE 18 (1-acetamido-3-methoxy-phenqzn-9J0-diyl) -cyanocobalamin The pre` edure and method of testing the aqueous extract de :'ibed in Example 15 is repeated except that a molar uivalent of l-acetamido-S-methoxy-phenazine is used instead of the 2-nitrophenazine. The new cobalamin, (l-acetamido-3methoxyphenazin9,10 diyl) cyano-cobalamin is found to be ionophoretically neu tral (when the pH of the system is 2.5); and the mobility of the new cobalamin in the filter paper chromatographic system using sec-butanol, water, KCN, and KC104 is 0.13 that of 5,6 dimethylbenzimidazole-cyano-coba1amin. The new cobalamin stimulates the growth of Ochromonas malhamenss.

In an analogous manner, by substituting a molar equivalent of 3-amino-2-phenazinol or 2-bromophenazine in place of 2-nitrophenazinol in Example l5, the corresponding cobalamins, namely (3 amino 2 hydroxyphenazin-9,l0-diyl)cyano7cobalamin and (2-bromo-phenazin-9,l0-diyl)cyanocobalamin are formed.

Similarly, by following the procedure of Example l but substituting the precursor indicatul and employing Organism used in the fermentation operations Cobalamin synthesized (from P. arab- P. P. P. zeae Rpmto- P.peter precursor listed inoyu'm rubrum thoenii (ATCC saceum .vlmii in Table I) (ATCC (ATCC (ATCC 4964) (ATCC (ATCC Example No.

5,6-dlmethyl-benzimidazole 19 20 21 22 23 -methy1-benzimidol 24 25 26 27 28 29 30 31 32 33 34 35 Benzotriazole 36 37 38 39 40 41 -triuorornethylbenzimidazole. 42 43 44 46 4 6 4-bromo-6-methoxybenzimidazole. 47 48 49 50 51 2-hydroxy-phenazine 52 Qulnoxaline 53 'Ihe invention may be variously otherwise embodied within the scope of the appended claims.

What is claimed is:

1. A process for preparing a physiologically active cobala'min which comprises culturing a precursor-requiring vitamin B12-producing microorganism of the genus Propionibacterium selected from the group consistting of Proponibacterum arabnosum, Propionibacterium thoeni, Propom'bacterium rubrum, lProponbacterium zeae, Propionibacterum pentosaceum and Propiom'bacterum petersoni under substantially anaerobic conditions in a cobalamin precursor-containing nutrient medium wherein the precursor is selected from the group 18 consisting of a quinazoline, a phenazine, quinoxaline, benzotriazole and 2nitro4triliuoromethylaniline, and recovering the resulting physiologically active cobalamin.

2. The process of claim 1 wherein the precursor is 2- 5 nitro-4-triuoromethylani1ine.

3. The process of claim 1 wherein the precursor is a quinazolne.

4. The process of claim 1 wherein the precursor is a phenazine.

5. The process of claim 1 wherein the microorganism is Propionibacterum arabinosum.

References Cited in the le of this patent UNITED STATES PATENTS OTHER REFERENCES Darken: The Botanical Review, vol. 19, No. 2, February 1953, article pp. 90 to 130. Pages 108-109, 114- 117 relied on.

Bernhauer et al.: Angewandte Chemie, vol. 66, No. 24, Dec. 21, 1954, PP. 776-780.

Fantes: Biochem. Jour. 59, l (1955) pp. 79-82.

Ford et al.: Biochem. Jour., January 1955, vol. 59, No. l, pp. -93. 

1. A PROCESS FOR PREPARING A PHYSIOLOGICALLY ACTIVE COBALAMIN WHICH COMPRISES CULTURING A PRECURSOR-REQUIRING VITAMIN B12-PRODUCING MICROORGANISM OF THE GENUS PROPIONIBACTERIUM SELECTED FROM THE GROUP CONSISTING OF PROPIONIBACTERIUM ARABINOSUM, PROPIONIBACTERIUM THOENII, PROPIONIBACTERIUM RUBRUM, PROPIONIBACTERIUM ZEAE, PROPIONIBACTERIUM PENTOSACEUM AND PROPIONIBACTERIUM PETERSONII UNDER SUBSTANTIALLY ANAEROBIC CONDITIONS IN A COBALAMIN PRECUSOR-CONTAINING NUTRIENT MEDIUM WHEREIN THE PRECUSOR IS SELECTED FROM THE GROUP 